The adhesion of mammalian cells to the extracellular matrix is of fundamental importance in regulating growth, adhesion, motility and the development of proper cellular phenotype. This has implications for normal development, wound healing, chronic inflammatory diseases, and tumor metastasis. Evidence accumulated over the last several years suggests that the molecular basis for the adhesion of both normal and transformed cells is complex and probably involves several distinct cell surface molecules. Extracellular matrices consist of three types of macromolecules: collagens, proteoglycans and noncollagenous glycoproteins.
One noncollagenous adhesive glycoprotein of interest is laminin. Laminin is a high molecular weight (.about.850,000) extracellular matrix glycoprotein found almost exclusively in basement membranes. (Timpl et al., J. of Biol. Chem., 254: 9933-9937 (1979)) The basement membrane is an ubiquitous, specialized type of extracellular matrix separating organ parenchymal cells from interstitial collagenous stroma. Interaction of cells with this matrix is an important aspect of both normal and neoplastic cellular processes. Normal cells appear to require an extracellular matrix for survival, proliferation, and differentiation, while migratory cells, both normal and neoplastic, must traverse the basement membrane in moving from one tissue to another.
Laminin consists of three different polypeptide chains: B1 with 215,000 MW, B2 with 205,000 MW and A with 400,000 MW (Timpl and Dziadek, Intern. Rev. Exp. Path., 29: 1-112 (1986)) When examined at the electron microscopic level with the technique of rotary shadowing, it appears as an asymmetric cross, with three short arms 37 nm long, each having two globular domains, and one long arm 77 nm long, exhibiting a large terminal globular domain (Engel et al., J. Mol. Biol., 150: 97-120 (1981)). The three chains are associated via disulfide and other bonds. Structural data shows that laminin is a very complex and multidomain protein with unique functions present in specific domains.
Laminin is a major component of basement membranes and is involved in many functions. Laminin has the ability to bind to other basement membrane macromolecules and therefore contributes to the structural characteristics of basement membranes. Laminin has been shown to bind to type IV collagen (Charonis et al., J. Cell. Biol., 100: 1848-1853 (1985); Laurie et al., J. Mol. Biol., 189: 205-216 (1986)) exhibiting at least two binding domains (Charonis et al., i J. Cell. Biol., 103: 1689-1697 (1986) Terranova et al., Proc. Natl. Acad. Sci. U.S.A., 80: 444-448 (1983). Laminin also binds to entactin/nidogen (Timpl and Dziadek, supra and to basement membrane-derived heparin sulfate proteoglycan (Laurie et al., J. Mol. Biol., 189: 205-216 (1986). Laminin also has the ability to selfassociate and form oligomers and polymers. Yurchenco et al., J. Biol. Chem., 260: 7636-7644 (1985). Another important functional aspect of laminin is its ability to associate with cell surface molecular receptors and consequently modify cellular phenotype in various ways. A receptor for laminin with a molecular weight of about 68,000 has been observed in various cell types (Lesot et al., EMBO. J., 2: 861-865 (1983; Malinoff and Wicha, J. Cell. Biol., 96: 1475-1479 (1983). However, at least one other and maybe more cell surface receptors for laminin may exist. [See Timpl and Dziadek, supra; Horwitz et al., J. Cell. Biol., 101: 2134-2166 (1985)]. These might include sulfatides, gangliosides [Roberts et al., Proc. Natl. Acad. Sci. U.S.A., 82: 1306-1310 (1985)] or various proteins and proteoglycans. These cell surface molecules may be mediators for the various effects that laminin has on cells. It is known that laminin can directly promote cell adhesion and cell migration of various cell types ranging from normal and malignant mesenchymal cells such as fibroblast and endothelial cells, to various epithelial cells Timpl and Dziadek, supra. However, the exact domains of laminin involved in such processes are not well established yet. For example, it is known that a heparin binding site exists on the A-chain, in the globule of the long arm of laminin (Ott et al., Eur. J. Biochem., 123: 63-72 (1982). However, the exact amino acid sequence of the A-chain is not known and therefore the related oligopeptide can not be identified yet.
Recently, a laminin fragment having a binding domain for a cell receptor without having a binding domain for type IV collagen has been described. U.S. Pat. No. 4,565,789 to Liotta et al. The Liotta patent discloses laminin fragments obtained by digestion of laminin with pepsin or cathepsin G. More specifically, digestion of laminin with pepsin or cathepsin G produces Pl (M.sub.r 280,000) and Cl (M.sub.r 350,000) fragments, wherein the long arm of the molecule is removed and also the globular end regions of the short arms are altered. C1 and P1 fragments having similar molecular weights and binding capacities can also be obtained by digestion of laminin with plasmin and chymotrypsin. Laminin is also known to stimulate neurite outgrowth, a function that has been primarily assigned to the lower part of the long arm of laminin (Edgar et al., EMBO J., 3: 1463-1468 (1986)).
The functions that have been described above make laminin an important component of many diverse and clinically important processes such as cell migration, wound healing, nerve regeneration, tumor cell metastasis through vascular walls [Liotta, Am. J. Path., 117: 339-348 (1984); McCarthy et al., Cancer Met. Rev., 4: 125-152 (1985)], diabetic microangiopathy, and vascular hypertrophy due to hypertension. Laminin could also be used in various devices and materials used in humans. In order to better understand the pathophysiology of these processes at the molecular level, it is important to try to assign each of the biological activities that laminin exhibits to a specific subdomain or oligopeptide of laminin. If this can be achieved, potentially important pharmaceuticals based on small peptides producing specific functions of the native, intact molecule, can be synthesized. In order to do this, the exact amino acid sequence of the three laminin chains needs to be determined. Up to now, only the B1 chain has been published. Sasaki, Proc. Natl. Acad. Sci. U.S.A., 84: 935-939 (1987).
Therefore, a need exists to isolate and characterize the subset of peptides within the B1 chain which are responsible for the wide range of biological activities associated with laminin. Such lower molecular weight oligopeptides would be expected to be more readily obtainable and to exhibit a narrower profile of biological activity than laminin itself or the B1 chain thereof, thus increasing their potential usefulness as therapeutic or diagnostic agents.